Bactericidal effect of cationic hydrogels prepared from ...

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Title Bactericidal effect of cationic hydrogels prepared from hydrophilic polymers

Author(s) 柴田, 優輝

Citation 北海道大学. 博士(生命科学) 甲第14215号

Issue Date 2020-09-25

DOI 10.14943/doctoral.k14215

Doc URL http://hdl.handle.net/2115/80659

Type theses (doctoral)

File Information Yuki_Shibata.pdf

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

https://eprints.lib.hokudai.ac.jp/dspace/about.en.jsp

Doctoral Dissertation

Bactericidal effect of cationic hydrogels prepared from

hydrophilic polymers

（親水性高分子から調製したカチオン性ハイドロゲルの殺菌効果に関する研究）

By

Yuki Shibata

Supervisor: Takayuki Kurokawa,

Laboratory of Soft & Wet Matter,

Graduate School of Life Science, Hokkaido University

Sapporo 001-0021, Japan

September, 2020

2

CHAPTER 1: General introduction ................................................................. 5

1.1 References ................................................................................................... 11

CHAPTER 2: Material .................................................................................. 19

2.1 Materials ...................................................................................................... 19

2.2 Live/dead® baclightTM bacterial viability kit ......................................................... 21

2.3 Preparation of hydrogels ........................................................................................ 21

2.3.1 Contact angle of the water drop on hydrogel ............................................. 23

2.3.2 Attenuated total reflection (ATR) spectroscopy .......................................... 24

2.4 Preparation of bacterial strain .................................................................. 25

2.5 References ......................................................................................................... 26

CHAPTER 3: Time-lapse microscopic observation .................................... 28

3.1 Introduction ..................................................................................................... 28

3.2 Experiments ..................................................................................................... 28

3.2.1 Materials ......................................................................................................... 28

3.2.2 Method ............................................................................................................ 29

3.3 Results and Discussion ................................................................................. 30

CHAPTER 4: Assessment of the antibacterial effect of the hydrogel .. 36

4.1 Introduction ..................................................................................................... 36

3

4.2 Experiments ..................................................................................................... 37

4.2.1 Materials ......................................................................................................... 37

4.2.2 Method ............................................................................................................ 37


CHAPTER 5: Factors contributing to the antibacterial property of

cationic hydrogels .................................................................................................... 43

5.1 Introduction ..................................................................................................... 43

5.2 Experiments ..................................................................................................... 43

5.2.1 Materials ......................................................................................................... 43

5.2.2 Method of antibacterial test ........................................................................ 44

5.2.3 Measurement of the initial elastic modulus of the hydrogel ...................... 44

5.2.4 Area density of the hydrogel monomers ...................................................... 45

5.2.5 Determination of the water weight ............................................................... 46


5.4 References ......................................................................................................... 51

CHAPTER 6: Bactericidal effect of cationic polymers .......................... 53

6.1 Introduction ..................................................................................................... 53

6.2 Material ............................................................................................................. 53

6.2.1 Preparation of polymer ................................................................................. 53

4

6.2.2 Preparation of bacterial strain ..................................................................... 53

6.3 Method ............................................................................................................... 54

6.3.1 Antibacterial test ............................................................................................ 54

6.3.2 Standard curve ............................................................................................... 54

6.4 Result and discussion .................................................................................... 56

CHAPTER 7: Disruption of bacterial cell membrane by cationic

hydrogel ....................................................................................................................... 58

7.1 Introduction ..................................................................................................... 58

7.2 Calculation of the elastic energy of the gel ............................................ 60

7.3 Calculation of the interfacial free energy .............................................. 60

7.4 Ion binding energy in the contact area ................................................... 61

7.5 References ......................................................................................................... 64

CHAPTER 8: Summary of the dissertation .................................................. 65

List of Publications .................................................................................................. 67

Original papers related to doctoral dissertation ...................................................... 67

Presentation in conferences related to doctoral dissertation .................................. 67

Acknowledgement ................................................................................................... 68

5

CHAPTER 1: General introduction

Hydrogel is a material which forms a three-dimensional network using a highly

hydrophilic polymer as a main chain. Hydrogels change their charge depending on the

polymer they are composed of, and many studies have been conducted using their

properties.1-5. In addition, hydrogels are hydrated polymeric materials usually

comprising >90% water by weight. Hydrogels are used in products such as contact

lenses and form the basis of cellular structure.6-8 Furthermore, hydrogels have been

assessed for their various biomedical applications9-11 However, hydrogels are

susceptible to bacterial contamination owing to their high water content and application

in humid environments.

It is very important to prevent bacterial contamination, numerous studies have focused

on the antibacterial properties of hydrogels. Numerous recent studies have focused on

the incorporation of bactericidal substances including silver nanoparticles 12-15 and

chitosan 16-20 into hydrogels. At present, many materials with bactericidal effects have

been discovered, and their mechanisms are also diverse. Bactericidal mechanisms can

be broadly divided into two. One is the mechanism by which germicides enter bacteria

and inhibit their metabolism. An example is penicillin. Penicillin binds to and inhibits

the cell wall peptidoglycan forming enzyme (PBP)21,22. As a result, the bacterium is

6

unable to form a cell wall and the cell wall gradually becomes thinner. The difference in

osmotic pressure between the inner cytoplasm and the outer fluid causes the bacteria to

expand and eventually rupture. The other is a mechanism by which a hole is made in the

cell membrane of a bacterium by physical action to kill bacteria. In recent years, it has

been clarified that the wing of cicada has such a sterilization mechanism. Microscopic

examination of the cicada's wings revealed numerous nano-sized pillars on its surface.

When bacteria come into contact with the pillar, the pillar penetrates the bacteria. The

cell membrane is pulled by the pillars, which eventually rupture and kill the bacteria. 23-

26

Heavy use of microbicides that block metabolism in bacteria, such as penicillin, risks

the bacteria developing resistance to the drug. In fact, penicillin-resistant Streptococcus

pneumoniae (PRSP) was known as a bacterium which acquired resistance to

penicillin.27,28

PRSP has a mutation in the penicillin-binding protein that is the target of penicillin, so

penicillin does not work. In this way, drug-resistant bacteria make the drug ineffective by

mutating the drug's target protein or by creating a drug efflux pump.29 Infections with

drug-resistant organisms are difficult to treat and increase the risk of serious illness and

death. On the other hand, the physical sterilization mechanism such as nano pillar attracts

7

attention because of the merit that bacteria are difficult to acquire resistance.

Some fungicides do not enter the cell but act directly on the cell membrane. One of these

is biguanide fungicides. Biguanide has a cationic functional group and numerous studies

have focused on its bactericidal effect. 30-34 These groups bind to the phospholipid head

groups of the anion-charged plasma membrane through ionic interactions. Binding of

biguanide fungicides to phospholipids on the outer leaflets of the plasma membrane is

believed to impair membrane fluidity. The attached region gradually forms a domain and

then separates from the plasma membrane. Cytoplasm leaks from this site, eventually

killing the bacteria. Substances displaying their activity on the surface of plasma

membranes, such as biguanide bactericide, are not affected by drug-resistant bacteria with

resistance mechanisms through drug efflux pumps. 35,36

In this paper, we focused on the bactericidal mechanism of biguanide fungicides and

evaluated the bactericidal effect of hydrophilic cationic hydrogels contain numerous

cationic groups bound to the polymer. These groups bind with phospholipids in the

outer leaflet of the plasma membrane, forming a polyelectrolyte complex. Similar to

biguanide bactericide, we expected that the cell membrane is disrupted at its poorly

fluid domain formed by cationic hydrogels and the bacteria are killed. Hydrophilic

cationic hydrogels are environmentally compatible materials because they do not

8

contain sustained release bactericides. Furthermore, this substance would not result in

bacterial resistance, since mechanisms underlying its bactericidal action involve the

destruction of cell membranes externally and do not involve the expression of drug

efflux pumps.

In Chapter 2, we described the synthesis methods and compositions of the hydrogels

used in this study, the types and culture methods of the bacteria used in the sterilization

test.

In Chapter 3, we examined the time required to sterilize cationic hydrogels. The bacteria

were seeded on a hydrogel, and the survival of the bacteria was observed by time laps.

Before observation, the hydrogel was impregnated with a fluorescent dye (SYTO -9) that

could stain live bacteria and a fluorescent dye (Propidium iodide) that could stain dead

bacteria.37 The life and death of the bacterium can be distinguished by the fluorescence

microscope observation. One place of the hydrogel surface was time-lapsed observed by

fluorescence microscopy. The survival curve was prepared by calculating the survival rate

of the bacteria at every elapsed time. Agar was used as a negative control. As the result,

it was clarified that the cationic hydrogel showed the sterilization effect by contacting

9

with the fungus for a long time.

In Chapter 4, three substrates were prepared: cationic hydrogel, neutral hydrogel, and

agar, on which bacteria were seeded. After the substrate was placed in an incubator for a

certain period of time, the microscopical observation was performed to calculate the

survival ratio of the bacteria. The bactericidal effect of cationic hydrogels was evaluated

by comparing the survival ratio between substrates. Next, in order to clarify whether the

bactericidal effect observed over time was unique to PDMAPAA-Q gel, several cationic

hydrogels (Crosslinking agent concentration: 6 mol%) with different monomer species

were prepared and the same bactericidal tests were performed on the cationic hydrogels.

The results showed that the cationic hydrogels showed high bactericidal activity in spite

of their different chemical structures.

In Chapter 5, we examined which factors in cationic hydrogels were associated with

bactericidal effects. Copolymer hydrogels were formed from neutral and cationic

monomers and their bactericidal effects on E. coli were investigated. The hydrogel

having different physicochemical properties was prepared by adjusting the ratio of the

cationic monomer constituting the hydrogel and the concentration of the crosslinking

10

agent. In this study, the correlation between the initial elastic modulus, the areal density

of cationic monomer unit, and the water content and the mortality ratio of bacteria was

investigated. The results showed a strong correlation with the areal density of cationic

monomer unit.

In Chapter 6, we investigated whether PDMAPAA-Q has a bactericidal effect even in the

uncrosslinked polymer state. The results showed that the death ratio of E. coli in cationic

polymer solutions was less than 40%. Furthermore, the death ratio of E. coli in cationic

polymers was compared with that of E. coli on cationic hydrogels. As the result, it was

clarified that the cationic polymer made from the same monomer species showed high

bactericidal effect by crosslinking.

In Chapter 7, we calculated the elastic energy of cationic hydrogels and the interfacial

energy of bacterial cell membranes and discussed the bactericidal mechanism of cationic

hydrogels from these values. The results showed that the elastic energy increased as the

area of adhesion between the bacteria and the cationic hydrogel increased, eventually

exceeding the interfacial tension of the cell membrane. These results suggest that the

cationic hydrogel makes extensive contact with bacteria by ionic interactions, destroys

11

the cell membrane by the elastic energy generated there.

Finally, in Chapter 8, the conclusion of this research were summarized.

1.1 References

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Pierfrancesco, C. The effect of the surface charge of hydrogel supports on thermophilic

biohydrogen production. Bioresource Technology 2010, 101 (12), 4386–4394.

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negatively charged microenvironment of collagen hydrogels regulates the chondrogenic

differentiation of bone marrow mesenchymal stem cells in vitro and in vivo. J. Mater

Chem B. 2020, doi: 10.1039/d0tb00172d.

(3) Soltys-Robitaille CE,; Ammon, DM, Jr; Valint, PL, Jr,; Grobe, GL, 3rd The

relationship between contact lens surface charge and in-vitro protein deposition levels.

Biomaterials 2001, 22 (24), 3257–60.

(4) Tao Lin Sun,; Takayuki, K.,; Shinya, K.,; Abu, Bin, Ihsan,; Taigo, A.,; Koshiro,

S.,; Md. Anamul, Haque,; Tasuku, N.,; Jian, P. G. Physical hydrogels composed of

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polyampholytes demonstrate high toughness and viscoelasticity. Nature Materials 2013

12 (10), 932-937.

(5) Feng, L.; Tao Lin Sun; Tasuku, N.; Takayuki K.; Yu, Z.; Koshiro, S.; Abu Bin

Ihsan; Xufeng, L.; Honglei, G; Jian, P. G. Oppositely Charged Polyelectrolytes form

Tough, Self-healing and Rebuildable Hydrogels. Advanced Materials 2015 27 (17),

2722-2727.

(6) Chaudhuri, O.; Viscoelastic hydrogels for 3D cell culture. Biomaterials Sci.

2017 5, 1480.

(7) Dosh, R.H.; Jordan-Mahy, N.; Sammon C.; Le, C.L. Use of l-pNIPAM

hydrogel as a 3D-scaffold for intestinal crypts and stem cell tissue engineering.

Biomater. Sci. 2019 7, 4310.

(8) Hyland, L.L.; Twomey, J.D.; Vogel, S.; Hsieh A.H.; Yu, Y.B.; Enhancing

biocompatibility of D-oligopeptide hydrogels by negative charges. Biomacromolecules

2013 14, 406-412.

(9) Naahidi, S.; Jafari, M.; Logan, M.; Wang, Y., Yuan, Y.; Bae, H.; Dixon B.;

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Chen, P. Biocompatibility of hydrogel-based scaffolds for tissue engineering

applications. Biotechnol. Adv. 2017 35 (5), 530-544.

(10) Ye, Y.N.; Frauenlob, M.; Wang, L.; Tsuda, M.; Sun, T.L.; Cui, K.; Takahashi,

R.; Zhang, H.J.; Nakajima, T.; Nonoyama, T.; Kurokawa, T.; Tanaka S.; Gong, J.P.

Tough and Self‐Recoverable Thin Hydrogel Membranes for Biological Applications.

Adv. Funct. Mater. 2018 28 (31), 1.

(11) Drury J.L.; Mooney, D.J. Hydrogels for tissue engineering: scaffold design

variables and applications. Biomaterials 2003, 24, 4337-4351.

(12) Murali Mohan, Y.; Lee, K.; Premkumar T.; Geckeler, K.E. Hydrogel networks

as nanoreactors: A novel approach to silver nanoparticles for antibacterial applications

Polymer 2007 48 (1), 158-164

(13) Li, M.; Jiang, X.; Wang, D.; Xu Z.; Yang, M. In situ reduction of silver

nanoparticles in the lignin based hydrogel for enhanced antibacterial application.

Colloid Surface B. 2019, 177, 370-376.

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(14) Carmen, C. Pairas; Clare, S. Mahon; David, K. Smith Self-assembled

supramolecular hybrid hydrogel beads loaded with silver nanoparticles for antimicrobial

application. Chemistry A European Journal 2020, doi: 10.1002/chem.202001349.

(15) Fan, L.; Xie, J.; Zheng, Y.; Wei, D.; Yao, D.; Zhang, J.; Zhang, T. Antibacterial,

Self-Adhesive, Recyclable, and Tough Conductive Composite Hydrogels for

Ultrasensitive Strain Sensing. ACS Appl. Mater. Interfaces 2020 12(19), 22225-22236.

(16) Ding, F.; Nie, Z.; Deng, H.; Xiao, L.; Du Y.; Shi, X. Antibacterial hydrogel

coating by electrophoretic co-deposition of chitosan/alkynyl chitosan. Carbohydr.

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(17) Noppakundilograt, S.; Sonjaipanich, K.; Thongchul N. Syntheses,

characterization, and antibacterial activity of chitosan grafted hydrogels and associated

mica-containing nanocomposite hydrogels. J. Appl. Polym. Sci. 2013 127, 4927-4938

(18) Lim, S.-H.; & Hudson, S. M. Review of chitosan and its derivatives as antimi-

crobial agents and their uses as textile chemicals. Journal of Macromolecular Science,

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Part C: Polymer Reviews, 2003 43(2), 223–269.

(19) Li, P.; Poon, Y. F.; Li, W.; Zhu, H.-Y.; Yeap, S. H.; Cao, Y. A polycationic

antimicrobial and biocompatible hydrogel with microbe membrane suctioning ability.

Nature Materials 2011 10(2), 149–156.

(20) Rabea, E. I.; Badawy, M. E. T.; Stevens, C. V.; Smagghe, G.; Steurbaut, W.

Chitosan as antimicrobial agent: Applications and mode of action. Biomacro- molecules,

2003 4 (6), 1457–1465.

(21) P.M. Blumberg & J. Strominger Interaction of penicillin with the bacterial cell:

penicillin-binding proteins and penicillin-sensitive enzymes. Bacteriol. Rev., 1974 38 (3)

291-335.

(22) B.G. Spratt The mechanism of action of penicillin. Science Progress, 1978 65

(257) 101-128.

(23) Ting, D.; Nilofar, F.; Terje, S.; Baptiste, L.; Howard F. J. Cicada-inspired cell-

instructive

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nanopatterned arrays. Scientific Reports. 2014 4,7122.

(24) Mary, N. D.; Elena, I. L.; Luis, A. R.; Nicolas, V.; Alnert F. Y. Nanopatterned

polymer surfaces with bactericidal properties. Biointerphases. 2015 10 (2), 021010.

(25) Sergey, P.; Jafar, H.; Vladimir, A. B.; Hyaden, K. W.; Vi K. T.; The Hong, P. N.;

Veselin, B.; Christopher, J. F.; Gregory, S. W.; Jolanta A. W.; Russell, J. C.; Elena, P. I.

Biophysical Model of Bacterial Cell Interactions with Nanopatterned Cicada Wing

Surfaces. Biophysical journal 2013 104 (4), 835-840.

(26) Jenkins, J.; Mantell, C. N.; Gholinia, A.; Verkade, P.; Nobbs, A. H.; Su, B.

Antibacterial effects of nanopillar surfaces are mediated by cell impedance, penetration

and induction of oxidative stress. Nature communications 2020 11, 1626.

(27) Villanova, Pa. Methods for dilution antimicrobial susceptibility tests for

bacteria that grow aerobically. Approved standard M 7-A 3. National Committee for

Clinical Laboratory Standerds. 1993.

(28) Klugman, K. P. Pneumococcal resistance to antibiotics. Clin Microbiol Rev 3

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1990, 171-196

(29) Hasdemir, U. The role of cell wall organization and active efflux pump systems

in multidrug resistance of bacteria. Mikrobiyoloji Bulteni. 2007 41 (2), 309-327.

(30) Chawner, J.A. A comparative study of the bactericidal and growth inhibitory

activities of the bisbiguanides alexidine and chlorhexidine J. Appl. Bacteriol. 1989, 66,

243.

(31) Ikeda, T., Tazuke S.; Watanabe, M. Interaction of biologically active

molecules with phospholipid membranes. I. Fluorescence depolarization studies on the

effect of polymeric biocide bearing biguanide groups in the main chain. Biochim.

Biophys. Acta 1983 735 (3), 380

(32) Broxton, P.; Woodcock, P. M.; Gilbert, P. A study of the antibacterial activty of

some polyhexamethylene biguanides towards Escherichia coli ATCC 8739. J. Appl.

Bacteriol. 1983, 54, 345-353.

(33) Kramer, A.; Eberlein, T. Re-evaluation of polihexanide use in wound

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antisepsis in order to clarify ambiguities of two animal studies. J. Wound Care 2019

28(4), 246-255.

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antibacterial efficacy of Ti-Al-V alloys coated with the antimicrobial agent PHMB. Acta

Biomater. 2020, doi: 10.1016/j.actbio.2020.02.016.

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(36) Gilbert P.; Moore, L.E. Cationic antiseptics: Diversity of action under a

common epithet J. Appl. Microbiol. 2005 99 (4), 703-715.

19

CHAPTER 2: Material

2.1 Materials

Three types of cationic hydrogels, a neutral hydrogel, copolymer hydrogels of cationic

and neutral monomers, and an agar gel were used herein. The three types of cationic

hydrogels were polymerized from (3-acrylamidopropyl) trimethylammonium chloride

(DMAPAA-Q) (Tokyo Chemical Industry Co., Ltd.),

acryloyloxyethyltrimethylammonium chloride (DMAEA-Q) (Toagosei Co., Ltd.), and

3- (methacryloylamino) propyltrimethylammonium chloride (MPTC) (FUJIFILM Wako

Pure Chemical Corporation). The neutral hydrogel was polymerized from N, N-

dimethylacrylamide (DMAAm) (FUJIFILM Wako Pure Chemical Corporation). The

copolymer hydrogels were polymerized from DMAPAA-Q and DMAAm. N, N '-

methylenbis (acrylamide) (MBAA) (FUJIFILM Wako Pure Chemical Corporation) and

used as a cross-linker. α-Ketoglutaric acid (α-keto) (FUJIFILM Wako Pure Chemical

Co., Ltd.) was used as an initiator. The chemical structures of the three cationic

polymers and the neutral polymer PDMAAm are shown in Figure 1. The agar gel was

prepared using agar powder. (FUJIFILM Wako Pure Chemical Corporation).

20

(Figure. 1) Chemical structures of the polymers of the hydrogel used in this

study.

21

2.2 Live/dead® baclightTM bacterial viability kit

Bacteria were stained using a live/dead baclightTM bacterial viability kit(Molecular

probes, City of Eugene, USA.) 1-4 in accordance with the manufacturer’s instructions,

and their viability on the gel was assessed. The kit comprised two fluorochromes:

SYTO -9 and propidium iodide (PI). SYTO -9 penetrates the bacterial cell membrane

and fluorescently stains live bacteria green (em: 480 nm/ex: 500 nm). PI, which does

not permeate the cell membrane, does not fluorescently stain live bacteria. Thus, PI only

fluorescently stains dead bacteria with defects in the plasma membrane (em: 490 nm/ex:

635 nm). 5 These fluorochromes were added in physiological saline. Hydrogels were

immersed in physiological saline containing fluorescent dyes overnight before seeding.

2.3 Preparation of hydrogels

Hydrogels were prepared through radical polymerization of each monomer and

crosslinking agent. When the hydrogel was polymerized, the total monomer

concentration was set at 2 M. The concentration of crosslinking agent MBAA and

initiator α-keto, monomer, were 2-12 mol% and 0.1 mol% relative to that of the

monomer, respectively. To synthesize the hydrogel, a reaction cell was fabricated by

sandwiching a silicone spacer along the edge between two parallel glass plates. Silicone

22

spacers of 2–5-mm thickness were used herein. Hydrogels for antimicrobial testing

were prepared using 2-mm-thick silicone spacers. Hydrogels for the analysis of the

elastic modulus were prepared using 5-mm-thick silicone spacers. The monomer

solution prepared herein was poured into the cell and the cell was placed in a glove box

filled with argon. The cells were irradiated using a UV lamp (Wavelength, 365 nm;

Intensity, 4mW/cm2) for approximately 8 h. After polymerization, the hydrogel was

eliminated from the glass plate and immersed in pure water for 1 week to remove

residual substances in the hydrogel. After 1 week, the hydrogels were transferred to

physiological saline (0.9 wt%) and immersed for 3 d. The hydrogel was then autoclaved

in physiological saline (120 °C, 20 min). After sterilization in an autoclave, the

hydrogels for antimicrobial tests were cut into disks (Φ = 25 mm) and immersed in

physiological saline supplemented with SYTO9 and PI for 1 d. Hydrogels for the

assessment of the elastic modulus were immersed in physiological saline and sterilized

in an autoclave.

23

2.3.1 Contact angle of the water drop on hydrogel

The contact angle was measured for the PDMAPAA-Q hydrogel, PDMAAm hydrogel,

and composite hydrogel. The monomer concentration was 2 M, while that of the cross-

linking agent MBAA was 10 mol%. Of the polymers constituting composite hydrogel,

60% was a PDMAPAA-Q and 40% was a PDMAAm. Water droplets were dropped onto

these hydrogels on measuring the contact angle. Since the contact angles of all

hydrogels were within 20°~40°, these hydrogels were confirmed to be hydrophilic

(Figure.2).

(Figure. 2) Image and contact angle of the water drop on poly (3-acrylamidopropyl)

trimethylammonium chloride (PDMAPAA-Q) hydrogel (a), poly N, N-

dimethylacrylamide (PDMAAm) hydrogel (b), and Composite hydrogel (c).

24

2.3.2 Attenuated total reflection (ATR) spectroscopy

Furthermore, ATR measurements were carried out to confirm the presence of cationic

functional groups in hydrogels containing cationic polymers (Figure.3)6,7. The samples

used for this measurement was the same as that used in 5.2. The monomer concentration

of the hydrogel was adjusted to 2 M and the crosslinker concentration to 6 mol%.

The fraction of cationic monomers in copolymer hydrogel is 0.6.

(Figure. 3) Measurement of poly (3-acrylamidopropyl) trimethylammonium chloride

(PDMAPAA-Q) hydrogel, poly N, N-dimethylacrylamide (PDMAAm) hydrogel and

copolymer hydrogel through attenuated total reflection (ATR) spectroscopy.

Copolymer

25

2.4 Preparation of bacterial strain

Micrococcus luteus (M. luteus NBRC 3333) and Escherichia coli (E. coli K -12) are

used as Gram-positive and Gram-negative strains herein, respectively. Single colonies

of E. coli were inoculated in liquid Luria-Bertani (LB) medium (Nippon Pharmaceutical

Co., Ltd.) and incubated (37 °C) for 1 d. Furthermore, a single colony of M. luteus was

inoculated on LB medium for 1 d in a shaker incubator (30 °C, 190 rpm). On

incubation, the bacterial density (M. luteus and E. coli) of each bacterium was adjusted

to 107–108 CFU/ml by measuring the turbidity of the bacterial solution, using a

spectrophotometer. To eliminate the LB medium from each bacterial suspension, the

bacteria were precipitated in a centrifuge (4500 rpm, 10 min, KUBOTA Co., Ltd, Model

4000- ), and the supernatant was eliminated. The bacterial pellet was washed with

physiological saline and reprecipitated in a centrifuge (4500 rpm, 10 min) to eliminate

the supernatant. Washing was repeated thrice to completely eliminate the LB medium.

After washing, the bacterial suspension was concentrated 10-fold with physiological

saline to increase the bacterial density in the suspension.

26

2.5 References

(1) Boulos, L.; M. Prevost; B. Barbeau; J. Coallier; and R. Desjardins. LIVE/DEAD

BacLight: application of a new rapid staining method for direct enumeration of viable and

total bacteria in drinking water. J. Micro- biol. Methods 1999 37, 77–86.

(2) Stocks, S. M. Mechanism and use of the commercially available viability stain,

BacLight. Cytom. A 2004 61,189–195.

(3) Gasol, J. M.; U. L. Zweifel; F. Peters; J. A. Fuhrman; and A. Hagstrom.

Significance of size and nucleic acid content heterogeneity as measured by flow

cytometry in natural planktonic bacteria. Appl. Environ. Microbiol. 1999 65, 4475–4483.

(4) Sachidanandham, R.; K. Y. Gin; and C. L. Poh. Monitoring of active but non-

culturable bacterial cells by flow cytometry. Biotechnol. Bioeng. 2005 89, 24–31.

(5) Berney, M.; Hammes, F.; Bosshard, F.; Weilenmann H.U.; Egli, T. Assessment

and interpretation of bacterial viability by using the LIVE/DEAD BacLight kit in

combination with flow cytometry. Appl. Environ. Microbiol. 2007 73 (10), 3283-3290.

27

(6) Bhuyan, M. M.; Okabe, H.; Hidaka, Y.; Hara, K. Pectin-[(3-

acrylamidopropyl) trimethylammonium chloride-co-acrylic acid) hydrogel prepared by

gamma radiation and selectivelysilver (Ag) metal adsorption. J. Appl. Polym. Sci. 2017,

135, 45906.

(7) Zheng, H.; Feng, L. Effect of the Cationic Block Structure on the

Characteristics of Sludge Flocs Formed by Charge Neutralization and Patching.

Materials 2017, 10, 487.

28

CHAPTER 3: Time-lapse microscopic observation

3.1 Introduction

As shown in General Introduction, biguanide fungicides with cationic charges show

bactericidal activity by contacting the bacterial cell membrane through ionic

interactions. First, in this study, in order to investigate whether hydrogels with cationic

charge in the main chain have bactericidal effect, bacteria were seeded on cationic

hydrogels and their survival was time-lapse microscopic observation. The sterilization

time of the cationic hydrogels was determined by calculating the bacterial mortality rate

at each elapsed time and creating survival curves.

3.2 Experiments

3.2.1 Materials

Poly (3-acrylamidopropyl) trimethylammonium chloride (PDMAPAA-Q) hydrogel

was used as a cationic hydrogel for time-lapse microscopic observation. The

concentration of crosslinking agent MBAA and initiator α-keto, monomer, were 6 mol%

and 0.1 mol% relative to that of the monomer (2M), respectively. We used agar as a

negative control. Agar was shaped to the same shape as the hydrogel and then immersed

in saline containing a fluorescent dye for one day before being used for experiments.

29

3.2.2 Method

The gel was warmed in water at 45℃ before the experiment so that the solution on the

gel surface rapidly evaporates. One microliter of each bacterial suspension was seeded

on the surface of the warmed gel. The gel seeded with bacteria was placed on a glass

dish and observed using a fluorescence microscope equipped with an incubator and a

portion of the gel surface was observed with time. Gels seeded with E.coli were

incubated at 37℃ and those seeded with M. luteus were incubated at 30℃.

Photographs were obtained every 15 min. Live and dead bacteria were manually

enumerated in each image. The survival ratio of bacteria in the images was determined

from the number of live bacteria divided by the total number (live and dead) of bacteria

and then multiplied by 100. The Bacterial survival ratio at each timepoint was

determined and the survival curves of each bacteria were plotted using Origin Pro

software. Herein, bacteria were seeded on a nutrient-free substrate. Therefore, the

bactericidal properties of the base were evaluated under the condition that the bacteria

did not proliferate during the experiment. To eliminate the increased death ratio of E.

coli on cationic hydrogels owing to natural bacterial death, the death ratio of E. coli on

hydrogels was corrected using the death ratio of E. coli in the negative control.

30

3.3 Results and Discussion

Time-lapse imaging revealed that almost all E. coli cells on agar and cationic

hydrogels did not die in a short period (Figure 4). Although only a few E. coli cells on

agar displayed red fluorescence after 12h, E. coli cells on the cationic hydrogel

displayed increased red fluorescence with time, and numerous bacterial cells dies after

12 h. The survival ratio of E. coli on the PDMAPAA-Q hydrogel was determined from

the ratio of the number of green and red fluorescent bacteria in each image (Figure.5).

The survival ratio of E.coli on after correcting for natural death exponentially decreased

with time, following a regression pattern explained using equation (4).

y = Aexp(-t/τ)+y0 ......(4)

where y is the survival ratio, A is the proportional constant, t is the elapsed time (h), τ is

the characteristic sterilization time (h), and y0 is offset. This survival curve reveals that

the characteristic sterilization time (τ) of the cationic hydrogel against E. coli is

approximately 6 h. Furthermore, the survival ratio of M. luteus on the gels was

determined on the basis of the ratio of the number of green and red fluorescent bacteria

in each image (Figure.6). The survival ratio of M. luteus on the PDMAPAA-Q hydrogel

corrected with the negative control began to rapidly decrease 4 h after seeding. When

the fitting curve was plotted after 4 h using equation (4), the value of τ was

31

approximately 3 h (Figure. 7). Commonly used bactericides exert a bactericidal effect

over a short period by degenerating cell membranes or inhibiting bacterial metabolism.

Simultaneously, during time-lapse microscopic observation, the cationic hydrogel

displayed sterilizing effects by interacting with the bacteria for a long period. Since the

present cationic hydrogel does not contain the releasing component, the polymer of the

cationic hydrogel interacts with the outer leaflet of the bacterial plasma membrane

through long-term interactions, displaying a sterilization effect. The difference in the

survival ratio between E. coli and M. luteus may have resulted from differences in the

thickness of the cell wall between gram-negative bacteria (E. coli) and gram-positive

(M. luteus) bacteria. Gram-positive bacteria have a thick cell wall surrounding the

plasma membrane, which may have led to the 4-h delay in the interaction between the

cationic hydrogel and the bacterial plasma membrane.

32

(Figure. 4) Images of live/dead cells on time-lapse fluorescence microscopic

observation. Images (a)~(c) show Escherichia coli (E. coli) on agar and (d)~(f)

show E. coli on the poly (3-acrylamidopropyl) trimethylammonium chloride

(PDMAPAA-Q) hydrogel. (a) and (d) indicate 0 min, (b) and (e) indicate 6 h, and

(c) and (f) indicate 12 h after seeding.

33

(Figure. 5) Survival ratio of Escherichia coli (E. coli) on agar gel (a) and the poly

(3-acrylamidopropyl) trimethylammonium chloride (PDMAPAA-Q) hydrogel (b).

Survival ratio of E. coli on PDMAPAA-Q is normalized in graph (a). The

characteristic sterilization time (τ) of the PDMAPAA-Q gel to E. coli was estimated

as 6 h.

34

(Figure. 6) Images of live/dead bacteria on time-lapse fluorescence microscopic

observation. Images (a)~(c) show Micrococcus luteus (M. luteus) on agar. Images

(d)~(f) show M. luteus on the poly (3-acrylamidopropyl) trimethylammonium

chloride (PDMAPAA-Q) hydrogel. Images (a) and (d) indicate 0 min. Images (b)

and (e) indicate >5 h. Images (c) and (f) indicate >10 h.

35

(Figure. 7) Time-lapse observation of Micrococcus luteus (M. luteus) on agar (a)

and the poly (3-acrylamidopropyl) trimethylammonium chloride (PDMAPAA-Q)

hydrogel (b). Survival ratio of M. luteus on the PDMAPAA-Q hydrogel was

normalized in graph (a). Sterilization of the PDMAPAA-Q hydrogel to M. luteus

was delayed for 4 h and the characteristic sterilization time [τ] was 3 h.

Consequently, the sterilization time was 7 h.

36

CHAPTER 4: Assessment of the antibacterial effect of the hydrogel

4.1 Introduction

Time-lapse fluorescence microscopic observation revealed that the cationic hydrogel

PDMAPAA-Q displayed a sterilization effect upon long-term interaction with the

bacteria. Furthermore, we evaluated the effect of charge on the bactericidal properties of

the hydrogels.

In Chapter 4, a cationic hydrogel, a neutral hydrogel, and agar were prepared, on

which bacteria were seeded. The gels were allowed to stand in an incubator for a period

of time, after which the viability of the bacteria on the gel surface was counted by

fluorescence microscopy. The bactericidal effect of cationic hydrogels was evaluated by

comparing the mortality of bacteria on each gel. Furthermore, several cationic hydrogels

with different monomer species (crosslinker density: 6 mol%) were prepared and we

examined whether the bactericidal effects observed on time-lapse fluorescence

microscopy on E.coli are unique to the PDMAPAA-Q hydrogel.

37

4.2 Experiments

4.2.1 Materials

In this experiment, cationic and neutral hydrogels and agar were used in this

experiment. The cationic hydrogels prepared three kinds of cationic hydrogels with

different monomer species, respectively. Three kinds cationic monomers, DMAPAA-Q,

DMAEA-Q and MPTC, were used to prepare cationic hydrogels herein. Neutral

hydrogel was prepared from DMAAm monomer. The crosslinker densities of these

hydrogels were prepared at 6 mol%. Two species, E. coli and M. luteus, were used in

the sterilization test.

4.2.2 Method

During time-lapse microscopy, fluorescent dye-containing gels were warmed in warm

water at 45 ℃. To prevent the gel from drying out, a wet gauze was placed on a plastic

Petri dish and a cover glass was placed over it. The gel was placed on a cover glass and

1μl of a suspension of each bacterium was seeded on the gel surface. In this experiment,

two samples were prepared for each hydrogel: one was observed immediately after

bacterial inoculation and the other sample was observed after allowing it to stand in an

incubator for a certain period. Gels seeded with E.coli were placed in an incubator

38

(37 ℃) for 12 h. Gels seeded with M. luteus were placed in an incubator (30 ℃) for 7

h. Bacteria on the gel surface were observed through fluorescence microscopy at

Hokkaido University Nikon Imaging Center. Photographs were randomly obtained for

20 fields per gel. Live and dead bacteria were manually enumerated in each image. The

death ratio of the bacteria in the photographs was determined using formula (2), where

D is the number of dead bacteria and L is the number of live bacteria. To eliminate

outliers, the Smirnoff-Grubbs rejection test was performed for the bacterial death ratio.

The Bonferroni test was performed to assess significant differences in the bacterial

death ratio between the two groups. The bacterial death ratio in the images was

determined on the basis of the number of dead bacteria divided by the total number

(dead and live) of bacteria and multiplied by 100. As previously reported, bacteria were

seeded on a nutrient-free substrate. Therefore, the bactericidal properties of the base

were evaluated under the condition that the bacteria did not proliferate during the

experiment.

39


We evaluated the effect of charge on the bactericidal properties of the hydrogels.

Cationic hydrogel (crosslinker density: 6 mol%), agar, and a neutral hydrogel

(PDMAAm) (crosslinker density: 6 mol%) were prepared, and the antibacterial test was

performed. E. coli and M. luteus were seeded on the gel surfaces, and 20 spots on each

gel surface were immediately imaged through fluorescence microscopy after seeding

and after a certain period. The bacterial death ratio was determined on the basis of these

images. The contact duration between the bacteria and the gel was set to12 h for E. coli

and 7 h for M. luteus, based on the time-lapse observations. The Bonferroni test was

performed to determine significant differences between the two groups after a certain

period after seeding. The death ratio of E. coli was 33.6% for neutral hydrogels, 27.3%

for agar, and 86.3% for cationic hydrogels at 12 h after seeding each sample

(Figure.8(a)). The death ratio of E. coli on the neutral gel was slightly higher than that

upon natural death. However, on comparing the cationic hydrogel with the neutral

hydrogel, the cationic properties imparted a more prominent bactericidal effect in the

cationic hydrogel. In the case of M. luteus, the bacterial death ratio on cationic

hydrogels was significantly higher than that on agar gel (Figure.8(b)). (p<0.01)

Furthermore, several cationic hydrogels with different monomer species (crosslinker

40

density: 6 mol%) were prepared and we examined whether the bactericidal effects

observed on time-lapse fluorescence microscopy on E.coli are unique to the

PDMAPAA-Q hydrogel. In this study, the Dannett’s test was performed to assess

significant differences in the death ratio of E. coli on hydrogels against agar gel.

Consequently, the death ratio of E. coli on PDMAEA-Q and PMPTC hydrogels 15 min

after seeding significantly differed from that of agar (Figure.9). Moreover, after 12 h,

the death ratio of E. coli on all cationic hydrogels was significantly higher than that in

the negative control. These results indicate that cationic hydrogels exhibit high

bactericidal activity even though their chemical structures are different.

41

(Figure. 8) The death ratio of (a) Escherichia coli (E. coli) and (b) Micrococcus

luteus (M. luteus) on hydrogels and agar after a certain period. Error bars indicate

the standard deviation. Significant differences between each sample at 12 h (a) or 7

h (b) after seeding were determined using the Bonferroni test (p<0.01).

42

(Figure. 9) Death ratio of Escherichia coli (E. coli) on cationic hydrogels and agar

after a certain period. Error bars indicate the standard deviation. Cationic hydrogel

samples after each time point were compared with agar after the same period.

Significant differences were observed between the result of agar and other samples,

as confirmed via Dunnett’s test. (p<0.01)

43

CHAPTER 5: Factors contributing to the antibacterial property of

cationic hydrogels

5.1 Introduction

It was revealed that the bactericidal effect was shown by bringing the cationic

hydrogel into contact with the fungus in chapter 3 and chapter 4. In chapter 5, to

determine which parameter of cationic hydrogels contributes to its bactericidal effects,

copolymer hydrogels of cationic and neutral monomers were formed from DMAPAA-Q

and DMAAm, and their bactericidal effects on E. coli were investigated. Herein, we

focused on the effect of the initial elastic modulus, the areal density of the cationic

monomer unit, and the water content of these hydrogels on its bactericidal effects. On

adjusting the fraction of the cationic monomer constituting the hydrogels and the

concentration of the crosslinking agent, hydrogels with different physicochemical

properties were prepared. (Figure 10)

5.2 Experiments

5.2.1 Materials

The copolymer hydrogel used in this experiment was prepared from the cationic

monomer DMAPAA-Q and the neutral monomer DMAAm. The total monomer

44

concentration was set at 2 M. The concentration of crosslinking agent MBAA and

initiator α-keto, monomer, were 2-12 mol% and 0.1 mol% relative to that of the

monomer, respectively. E. coli was used for this antimicrobial test.

5.2.2 Method of antibacterial test

Antimicrobial testing was performed in the same manner as in 4.2. 2.

5.2.3 Measurement of the initial elastic modulus of the hydrogel

The initial elastic moduli of these hydrogels were determined through indentation

tests. The force -displacement relationship for each hydrogel was determined using a

universal mechanical test apparatus (Tensilon RTC -1310 A: Orientech.) equipped with

a hemispherical steel indenter with a 2-mm radius.

The initial elastic modulus (E) of the hydrogel was determined using Equation (1)

derived from Hertz's equation. 1-4

𝑓 𝐸 𝑙 ・・・(1)

Where, f, l, R, and 𝑣 are the measured force, indenter displacement, indenter radius,

and the Poisson’s ratio of the hydrogel, respectively. The 𝑣 was 0.5 assuming that the

hydrogel volume remains unchanged during the indentation test. The value of E was an

45

average of 5 measurements. (Figure. 11 (a))

5.2.4 Area density of the hydrogel monomers

The area densities of the hydrogel monomers were determined using the following

method. Upon radical polymerization of the hydrogel, the hydrogel thickness t0 was

measured thrice and the average value was determined. The hydrogel was then swollen

with physiological saline. Thereafter, the hydrogel was immersed in physiological saline

and sterilized in an autoclave (120℃, 20 min). Thereafter, the hydrogel thickness t was

determined thrice and the average value was determined. The volume swelling ratio

q=(t/t0)3 was determined on the basis of the changes in thickness, and the "Area density

of the monomer" and "Areal density of cationic monomers" s of the hydrogels were

determined on the basis of the swelling ratio q and initial monomer concentration C0 in

feed, (Figure. 11 (b))

s=(C0/q)2/3 ...(2)

46

5.2.5 Determination of the water weight

The water weight of the hydrogels after swelling in saline and sterilization in an

autoclave was determined as follows

𝑊𝑎𝑡𝑒𝑟 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 % 1 ∗ 100 ...(3)

where q is the volume swelling ratio of the hydrogels swollen in saline, ds is saline

density (1.007 g/ml), dg is density of the as-prepared gel, and cp is the polymer

concentration (g/ml) in the hydrogel prepared herein. For dg and cp, we assume that dg

and cp were approximately equal to the monomer density and concentration (g/ml) of

the precursor solutions of the hydrogels, respectively. (Figure. 11 (c))

47

(Figure. 10) The death ratio of (a) Escherichia coli (E. coli) on copolymer

hydrogels. Error bars indicate the standard deviation.

48

(Figure. 11) Correlation between cross-linker concentration, fraction of the cationic

monomer, and physicochemical properties of copolymer hydrogels prepared from

(3-acrylamidopropyl) trimethylammonium chloride (DMAPAA-Q) and N, N-

dimethylacrylamide (DMAAm). (a) Initial elastic modulus of the hydrogels. (b)

Areal density of the cationic monomer of the hydrogels. (c) Weight water content of

the hydrogels.

49


The factors influencing the bactericidal effect of the cationic hydrogels were

investigated by plotting the death ratio of E. coli. against the initial elastic modulus

(Figure 12(a)), the water weight of the hydrogels (Figure 12(b)), and the areal density of

the cationic monomers of the hydrogels (Figure 12(c)). The bacterial death ratio was not

significantly correlated with the initial modulus and water content of the hydrogels;

however, it was strongly correlated with the areal density of the cationic monomers, i.e.,

higher the areal density of the cationic monomers, stronger the bactericidal effect of the

cationic hydrogels. Previous studies have suggested that hydrophilic cationic polymers

share ionic interactions with plasma membrane molecules, and the incorporation of

membrane molecules with the polymers disrupts the membrane assembly.5,6 Indeed,

studies on hydrogels and protein adsorption have reported that cationic hydrogels

adsorb large amounts of anionic charged proteins. 7

50

(Figure. 12) Death ratio of Escherichia coli (E. coli) on hydrogels as a function of

the physicochemical properties of the hydrogel. (a) Initial modulus, (b) water

weight of the hydrogels, (c) areal density of the cationic monomer of the hydrogels.

Symbol ■ indicates hydrogels containing cationic polymers. Symbol ▲ indicates

hydrogels containing only neutral polymers (zero fraction of the cationic monomer).

R is the correlation coefficient.

51

5.4 References

(1) Hoshino, K.I.; Nakajima, T.; Matsuda, T.; Sakai T.; Gong, J.P. Network

elasticity of a model hydrogel as a function of swelling ratio: from shrinking to extreme

swelling states. Soft Matter 2018 14, 9693.

(2) Y. Hu; X. Zhao; J. J. Vlassak and Z. Suo Using indentation to characterize the

poroelasticity of gels. Appl. Phys. Lett., 2010 96, 121904.

(3) C. T. McKee; J. A. Last; P. Russell and C. J. Murphy Indentation Versus Tensile

Measurements of Young's Modulus for Soft Biological Tissues. Tissue Eng., Part B, 2011

17 (3), 155-164.

(4) D. C. Lin; D. I. Shreiber; E. K. Dimitriadis and F. Horkay Spherical Indentation

of Soft Matter Beyond the Hertzian Regime: Numerical and Experimental Validation of

Hyperelastic Models. Biomech. Model. Mechanobiol., 2009 8 (5), 345-358.

(5) Li, P.; Poon, Y.F.; Li, W.; Zhu, H.Y.; Yeap, S.H.; Cao, Y. Qi, X.; Zhou, C.;

Lamrani, M.; Beuerman, R.W.; Kang, E.T.; Mu, Y.; Li, C.M.; Chang, M.W.; Jan Leong

52

S.S.; Chan-Park, M.B. A polycationic antimicrobial and biocompatible hydrogel with

microbe membrane suctioning ability. Nat. Mater. 2011, 10 (2), 149-156.

(6) Gabriel, G. J.; Som, A.; Madkour, A. E.; Eren, T. & Tew, G. N. Infectious

disease: Connecting innate immunity to biocidal polymers. Mater. Sci. Eng. R 2007 57,

28–64.

(7) Guo H.; Uehara Y., Matsuda T.; Kiyama R.; Li L.; Ahmed J.; Katsuyama Y.;

Nonoyama T.; Kurokawa T. Surface charge dominated protein absorption on hydrogels

Soft Matter 2020, 16, 1897

53

CHAPTER 6: Bactericidal effect of cationic polymers

6.1 Introduction

Previous experiments revealed that the bactericidal effect of cationic hydrogels is

important to have cationic functional groups regardless of the monomer species.

In Chapter 6, we investigated whether PDMAPAA-Q has a bactericidal effect even in the

uncrosslinked polymer state.

6.2 Material

6.2.1 Preparation of polymer

In this chapter, two types of polymers were used: PDMAPAA-Q as a cationic polymer

and PDMAAm as a neutral polymer. Each monomer solution was prepared with a

monomer concentration of 2 M and an initiator concentration of 0.1 mol% and

subjected to UV polymerization in an argon atmosphere for 8 hours. The polymer

solution was dialyzed with stirring in pure water for 5 days using a semipermeable

membrane to remove unpolymerized monomers. They then used a vacuum oven to

remove the moisture from the polymer solution and pulverized it into a polymer powder

for use in the experiment.

6.2.2 Preparation of bacterial strain

In this experiment, E. coli was used as the species. The bacterial suspension was prepared by 2.4

methods.

54

6.3 Method

6.3.1 Antibacterial test

In this chapter, after sterilization testing, E. coli was stained with a fluorescent dye

and mortality of E. coli was measured using a fluorospectro-photometer. First, the

polymer powder was added in physiological saline at concentrations of 1 M, 0.75 M,

0.5 M, and 0.25 M, and sterilized in an autoclave. Thereafter, the prepared fungal

solution was added to the polymer solution and left in an incubator (37 ° C) with stirring

for 12 hours. Saline was then added to the solution to reduce the viscosity, and a

fluorescent dye was added to stain E. coli. The death ratio of E.coli was calculated using

a fluorospectro-photometer and a calibration curve.

6.3.2 Standard curve

After incubation of E. coli in LB liquid medium, the culture is divided into two parts.

Both were washed with saline and one was sterilized with 70% ethanol. One bacterial

fluid contains only live E. coli and the other bacterial fluid contains only dead E. coli.

By mixing these two kinds of bacterial solutions, a bacterial solution containing 100%,

75%, 50%, 25% and 0% of dead E. coli was prepared and stained with a fluorescent

dye. Fluorescence emission of the bacterial solution was measured by fluorospectro-

photometer, and the areas of wavelength between 490 nm and 520 nm (Area (1)) and

55

600 nm and 630 nm (Area (2)) were calculated. Area (1) is the fluorescence emitted by

live E. coli, and area (2) is the fluorescence emitted by dead E. coli. The standard curves

were obtained by dividing the area of Area (1) by the area of Area (2). (Figure.13)

(Figure. 13) Standard curve of E. coli. The horizontal axis shows the percentage of

viable E.coli. The vertical axis shows the value obtained by dividing Area (1) by

Area (2).

0

1

2

3

4

5

6

7

8

0% 20% 40% 60% 80% 100% 120%

Green

/red

fluorescen

ce

Ratio of live E.coli(%)

56

6.4 Result and discussion

Most samples of cationic and neutral polymers showed less than 40% death ratio against

E.coli. (Figure.14) Furthermore, the death ratio of E. coli in cationic polymers was

compared with that of E. coli on cationic hydrogels. (Figure.15) The data of the cationic

hydrogel used at this time were 1.0 samples of fraction of cationic monomer in Figure

11(b). As the result, it was clarified that the cationic polymer made from the same

monomer species showed high bactericidal effect by crosslinking. The bactericidal

mechanism of biguanide fungicides mentioned in the introduction is thought to be

important to fix and inhibit cell membrane fluidity. It is considered that the cationic

polymer can inhibit the flow of cell membrane more strongly than the polymer state by

gelation with a crosslinking agent.

57

(Figure. 15) Death ratio of E.coli in cationic polymer solution and on cationic

hydrogel.

(Figure. 14) Death ratio of E.coli in cationic polymer solution (PDMAPAA-Q) and

neutral polymer solution (PDMAAm)

58

CHAPTER 7: Disruption of bacterial cell membrane by cationic

hydrogel

7.1 Introduction

From previous chapters, it has been suggested that contact between cationic hydrogels

and bacterial cell membranes via ionic interactions is important for bactericidal effects.

In Chapter 5, the mechanism by which hydrogels are sterilized after contact with bacteria

is discussed based on the physical properties of hydrogels. In this study, we focused on

the elastic energy of the gel resulting from local deformation at ion-binding sites between

the bacteria and hydrogels. The deformation of the hydrogel due to drying and the

movement of the bacteria generate elastic energy at the ion-binding sites of the bacteria

and the hydrogel. We considered that when elastic energy (Wel) exceeds the interfacial

free energy (Wf) required to expose the hydrophobic ends of the lipid bilayer in solution,

the lipid bilayer is considered to have been disrupted (Figure.16). In Chapter 5, it was

discussed whether the theory of this sterilization mechanism was realistic by actually

calculating elastic energy and comparing it with the interfacial tension of cell membrane.

59

(Figure. 16) Schematic representation of the mechanism underlying the sterilization

effect of the cationic hydrogels, involving the atrial elimination of lipid bilayers

from the bacterial cell membranes.

60

7.2 Calculation of the elastic energy (Wel) of the gel

Wel (4) were estimated from the following equations. When calculating the local elastic

energy, the deformation region of the hydrogel in which it occurs was assumed to be a

sphere. The elastic energy is increased not only by the magnitude of the strain in the ion-

binding region of the hydrogel and the bacterium but also by the expansion of the ion-

binding region.

𝑊 𝐸𝜀 ∗ 𝜋𝑟 ...(4)

Where, E, ε and r are the Young’s modulus of the PDMAPAA-Q gel (monomer

concentration, 2 M; cross-linker concentration, 6 mol%), the strain, and the radius of the

contact area (m), respectively. Values of ε were set from 0.06 to 0.1.

7.3 Calculation of the interfacial free energy (Wf)

Wf (5) were estimated from the following equations.

𝑊 2 𝛾𝑡2𝜋𝑟 ...(5)

Where, γ and t are the interfacial tensions of n-heptadecane (53.2N/m2) 1,2 and the

length of the hydrophobic portion of a single phospholipid molecule that forms the

bacterial lipid bilayer(2.13nm), respectively. The interfacial energy (Wf) determined

herein was doubled because two hydrophobic regions (r) were formed upon detachment

of the cell membrane. The Wf calculated by this equation was the value required to

61

remove the entire lipid bilayer structure where the cell and hydrogel are bound to ionic

interactions from the cell membrane. The reason why the whole bilayer structure was

chosen as the target for the destruction of the plasma membrane was that it requires less

energy to pull out the entire bilayer structure than to pull out only mono layer of the

lipid bilayers.

7.4 Result and discussion

A graph of r on the horizontal axis and W on the vertical axis was plotted on the basis

of equations (4) and (5) (Figure.17). The graph shows that Wel exceeds Wf when r

increases and exceeds a certain value. With an increase in ε, Wel exceeds Wf in a smaller

adsorption area. In other words, the membrane can be disrupted with an adsorption

radius of several tens of nanometers. These findings indicate that cationic hydrogels

strongly bind to bacteria through their cationic charges, and the contact area between the

bacteria and the hydrogel was gradually increased. This ion-binding region was

considered to have been eliminated from the bacterial cell membrane owing to certain

factors.

62

(Figure. 17) Dependence of cell membrane interfacial energy and hydrogel elastic

energy [W (J)] on adsorption radius between the bacteria and the hydrogel [r (m)].

63

7.5 Ion binding energy in the contact area

It was clarified that the elastic energy (Wel) exceeded The interfacial energy (Wf) in the

realistic value in chapter 7.4. Next, we examined whether the energy of the ionic bond

of the contact region between the cell membrane and cationic hydrogel exceeded Wel

when the cell membrane was pulled out by cationic hydrogel. The value of the energy of

the ionic bond in physiological saline solution (Ea=1.7E-20(J)) was cited from the

reference3.

From figure 17, when the strains were 0.06 and 0.1, the contact area was calculated

using the adsorption radius (r) at which Wel exceeded Wf. The values of areal density of

the cationic monomer unit from Figure 10 (b) were used. From r and the values of areal

density of the cationic monomer unit, the number of cationic monomer unit in the

contact area was calculated. Then, the energy of the ionic bond in the contact area was

calculated from the value of the energy of the ionic bond in the physiological saline and

the number of cationic monomer unit.

As a result, when the strain was 0.06, the ion binding energy in the contact area when

Wel exceeded Wf was 4.3 E -15 (J). This value was found to be stronger than Wel (5.2E-

16 J) in this condition. when the strain was 0.1, the ion binding energy in the contact

area when Wel exceeded Wf was 1.5 E -15 (J). This value was also stronger than Wel

64

(5.2E-16 J). This suggests that the ionic bond does not break before the hydrogel

abstracts the cell membrane.

7.6 References

(1) Rehfeld, S.J. Adsorption of sodium dodecyl sulfate at various hydrocarbon-

water interfaces J. Phys. Chem. 1967 71, 738-745.

(2) Tuan, V.; Vu, D. Synergistic effects of surfactants and heterogeneous

nanoparticles at oil-water interface: Insights from computations. Journal of Colloid and

Interface Science. 2019 553 (1), 50-58.

(3) Evan, S.; Sebastiaan, A. Direct Measurement of the Strength of Single Ionic

Bonds between Hydrated Charges. ACS. NANO, 2012,6, (6), 5297-5303.

65

CHAPTER 8: Summary of the dissertation

In this paper, to investigate the bactericidal activity of cationic hydrogel, bacteria were

seeded on the cationic hydrogel, and their life and death were observed by time lapse.

When the characteristic sterilization time of the cationic hydrogel was calculated by

making the survival curve of the bacterium, the bactericidal time was about 6 hours for

the gram-negative bacterium and about 7 hours for the gram-positive bacterium. It was

clarified from this result that the sterilization effect was shown by the contact with

bacteria for a long time in the cationic hydrogel.

In chapter 4, we evaluated the bactericidal effects of cationic hydrogels. Cationic

hydrogel, neutral hydrogel, and agar which was negative control were prepared, on which

bacteria were seeded and allowed to stand for a fixed time. When the survival ratio of the

bacteria was compared afterwards, the cationic hydrogel showed high bactericidal effect.

In chapter 5, we examined the relationship between the physical properties of hydrogels

and their bactericidal effects. A plurality of composite hydrogels prepared from cationic

monomers and neutral monomers having different physical properties were prepared, and

their sterilization effects were evaluated. The areal density of the cationic monomers

showed a high correlation with the Death ratio of bacteria. We considered that ionic

bonding by cationic charge of hydrogel and anionic charge of cell membrane plays an

important role in bactericidal effect.

66

Furthermore, in Chapter 6, the bactericidal effect of cationic polymers was measured

and compared with that of cationic hydrogels, suggesting that gelation with a crosslinking

agent enhances the bactericidal effect.

Next, the mechanism of sterilization after ion binding was discussed from the

viewpoint of elastic energy generated in the binding region and interfacial tension of the

cell membrane. As a result, it was clarified that the the elastic energy (Wel) increased as

the binding region between the cell membrane and hydrogel increased or the strain

increased and exceeded the interfacial energy (Wf) of the cell membrane at a realistic

value. When the Wel exceeds the Wf, the ion binding region is pulled out from the cell

membrane, and the cell membrane is destroyed.

In this paper, it was shown that the sterilization effect could be easily given to the

hydrogel by using the cationic monomer. And, it is considered that a large contribution

can be made to future research on sterilization by examining the sterilization mechanism

from the viewpoint of the elastic energy which is not conventional.

67

List of Publications

Original papers related to doctoral dissertation

(1) Yuki Shibata, Takayuki Kurokawa, Tomoyasu Aizawa, Jian Ping Gong:

“Bactericidal effect of cationic hydrogels prepared from hydrophilic polymers”

Journal applied polymer sci 誌、Accepted

Presentation in conferences related to doctoral dissertation

(1) Yuki Shibata, Takayuki Kurokawa, Tomoyasu Aizawa, Jian Ping Gong ：

“Antibacterial property of cationic hydrogel” Gelsympo 2018, 4dMS (日本)

68

Acknowledgement

This research on this dissertation has been carried out in the Laboratory of Soft and Wet

Matter (LSW), Graduate School of Life Science, Hokkaido University, Japan, supervised

by Professor Takayuki Kurokawa. Associate examiners are Professor Jian Ping Gong,

Professor Tomoyasu Aizawa.

Firstly, I greatly appreciate all the support and encouragement of my respected

supervisor, Professor Takayuki Kurokawa during master’s and doctoral study in LSW. He

gave me not only tremendous academic support to be an outstanding researcher but also

so many wonderful opportunities. I really appreciate that he evaluated my doctoral

research, and through a lot of discussions with him, I could acquire a deep understanding

of polymer materials and an ability to perform research. I really respect his positive

attitude, research understanding and considering.

I would like to many thanks also to Professor Jian Ping Gong, Professor

Tomoyasu Aizawa, Associate Professor Tasuku Nakajima, Assistant Professor Takayuki

Nonoyama for their helpful scientific comments, discussions and suggestions regarding

my research. I am also very grateful to post-doctoral fellows, guest researcher and

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research assistants in “LSW”. I also would like to thank the staffs in LSW; Ms. Eiko

Hasegawa, Ms. Yuki Okubo, Ms. Kato and Ms. Iseya for helping me in financial works

and lab life of Hokkaido University.

Finally, special thanks to my family for all the support and encouragement during

the life in Hokkaido University. I dedicate this dissertation to them.

Yuki Shibata

Laboratory of Soft & Wet Matter

Graduate School of Life Science

Hokkaido University

Sapporo Japan

September 2020

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